Osteopontin-related compositions and methods

ABSTRACT

This invention provides a method for reducing the amount of osteopontin in an osteopontin-expressing cell comprising introducing into the cell a nucleic acid which specifically inhibits osteopontin expression in the cell. This invention also provides methods for inhibiting the onset of, and treating, osteopontin-related disorders, as well as compositions for practicing the same. This invention further provides methods for determining the amount of osteopontin in a sample, and a kit for practicing the same. This invention also provides methods for determining whether an agent reduces the amount of osteopontin in an osteopontin-expressing cell. Finally, this invention provides methods for treating a subject afflicted with a disorder mediated by an endogenous protein.

The invention disclosed herein was made with Government support underGrant No. R0118235 from the National Institutes of Health of the UnitedStates Department of Health and Human Services. Accordingly, the UnitedStates Government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a US national stage of PCT/US02/37466, filedNov. 21, 2002, which claims the benefit of U.S. Ser. No. 60/332,071,filed Nov. 21, 2001, both of which are incorporated by reference for allpurposes.

Throughout this application, various publications are referenced inparentheses. Full citations for these publications may be found listedat the end of the specification. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art asknown to those skilled therein.

FIELD OF THE INVENTION

This invention relates to treating and preventing osteopontin-relateddisorders such as multiple sclerosis, and quantitatively measuringosteopontin in a sample. This invention also relates to methods of usingosteopontin in the treatment of disorders, such as autoimmune disorders,that are mediated by endogenous proteins.

BACKGROUND OF THE INVENTION

Multiple Sclerosis

Multiple sclerosis (“MS”) is a demyelinating disease characterized byinflammation in the brain and spinal cord. MS is the most common humanautoimmune disease involving the nervous system. In the United States,approximately 250,000 individuals suffer from MS. In MS, cells of theimmune system invade and destroy myelin, the fatty material thatinsulates nerves in the brain and spinal cord. Other CNS cells produce ahardened sclerotic lesion (plaque) around the multiple demyelinatedsites. Neurologic findings suggest lesions in separate areas of the CNSthat occur at different times.

A typical presentation of MS involves an initial course, running forseveral years to more than a decade, manifest by episodes of relapsefollowed by remission. Relapses often follow an episode of a viralinfection of the upper respiratory system or gastrointestinal tract. Inabout one third of MS patients, this disease evolves into a progressivecourse termed “secondary progressive MS.” In a minority of patients,progressive neurologic deterioration without remission occurs from theonset of disease, and this is called “primary progressive MS.” Thepathophysiologic and genetic causes underlying primary versus secondaryprogressive MS remain unclear.

Clinical problems observed in MS patients may include disturbances invisual acuity, sometimes culminating in blindness; double vision; motordisturbances affecting walking and use of the hands; uncoordination;bowel and bladder incontinence; spasticity; and sensory disturbancesincluding loss of touch, pain, temperature and proprioception. Thepathology of MS lies entirely in the central nervous system and ischaracterized by a classic picture of inflammation surrounding venulesand extending into the myelin sheath.

Immune responses to various components of the myelin sheath have beendetected in MS patients. These components include myelin basic protein(“MBP”), proteolipid (“PLP”), transaldolase and 2′,3′ cyclic nucleotide3′phosphodiesterases (“CNP”), as well as two members of theimmunoglobulin supergene family found in the myelin sheath, i.e., myelinoligodendroglial glycoprotein (“MOG”) and myelin-associated glycoprotein(“MAG”) (11). In addition, some inducible heat shock proteins, includingcrystallin-B, can be detected in glial cells in MS lesions and canstimulate an immune response in MS patients. The major T and B cellresponse in the central nervous systems of the roughly two thirds of MSpatients who are HLA DR2 is directed to a region between residues 84 and103 of MBP (14, 18).

Osteopontin

Osteopontin (“OPN”), also called early T cell activation gene-1, is ahuman protein whose primary structure has been characterized (25). OPNis a pleiotropic protein having a conserved RGD binding motif, and whenproduced by osteoblasts is involved in the anchoring of osteoclasts tothe mineral of bone matrix. Expression of osteopontin in bone tissue isstimulated by 1-alpha-1,25-dihydroxyvitamin D3. Osteopontin alsoprovides the protein matrix for urinary stones.

Osteopontin also plays roles in inflammation and in immunity toinfectious diseases (29). Osteopontin costimulates T cell proliferation(8), and is classified as a Th1 cytokine, due to its ability to enhanceIFN-gamma and IL-12 production, and to diminish IL-10 (32). It has beenshown that osteopontin expression in rat aortic smooth muscle cells isinhibited by NK-104, a 3-hydroxy-3-methylglutaryl coenzyme A reductaseinhibitor.

SUMMARY OF THE INVENTION

The invention provides methods for preventing or treating a patientsuffering or at risk of a disorder in which expression of osteopontincontributes to the pathogenesis. These methods comprise administering tothe subject an effective amount of a nucleic acid comprising a segmentencoding osteopontin, whereby the nucleic acid is expressed in thepatient to produce osteopontin, and the osteopontin induces an immuneresponse that reduces the level of osteopontin in the patient. In somemethods, the immune response includes formation of antibodies. In somemethods, the patient is suffering from or at risk of raft versus hostdisease, epilepsy, a granulomatous disorder, herpes simplex keratisits,bacterial arthritis, or an autoimmune disease. Examples of autoimmunediseases include multiple sclerosis, rheumatoid arthritis, type Idiabetes.

The nucleic acid can be DNA, in which case the nucleic acid furthercomprises a promoter and optionally an enhancer in operable linkage tothe segment encoding the osteopontin. The promoter can be constitutiveor cell-type specific. Alternatively, the nucleic acid can be RNA. Insome methods, the nucleic acid is administered intramuscularly. In somemethods, the the subject is a human.

Some methods, further comprise monitoring a decrease in the level ofosteopontin responsive to the administering step. In some methods, thelevel of osteopontin is monitored in a cell of the patient selected fromthe group consisting of a neuron, a macrophage, a vascular endothelialcell, an astrocyte and a microglial cell. In some methods, the patienthas the disorder and the method further comprises monitoring a decreasein the symptoms of the patient responsive to the administering.

The invention further provides a composition comprising a nucleic acidencoding osteopontin and a pharmaceutically acceptable carrier.

The invention further provides methods for preventing or treating apatient suffering from or at risk of a disorder in which expression ofosteopontin contributes to the pathogenesis. These methods compriseadministering to the patient an effective amount of osteopontin, wherebythe osteopontin induces an immune response that reduces the level ofosteopontin in the patient. In some such methods, the the osteoponin isadministered with an adjuvant. In some methods, the immune responsecomprises formation of antibodies to osteopontin.

In some methods, the patient is suffering from or at risk of graftversus host disease, epilepsy, a granulomatous disorder, herpes simplexkeratisits, bacterial arthritis or an autoimmune disease as describedabove. In some methods the patient is a human.

Some methods further comprise monitoring a decrease in the level ofosteopontin responsive to the administering step. In some methods, thelevel of osteopontin is monitored in a cell of the patient selected fromthe group consisting of a neuron, a macrophage, a vascular endothelialcell, an astrocyte and a microglial cell. In some methods, the thepatient has the disease and the method further comprises monitoring adecrease in the symptoms of the patient responsive to the administering.

The invention further provides a composition comprising osteoponin andan adjuvant.

This invention provides a method for reducing the amount of osteopontinin disorders in which osteopontin is produced.

This invention also provides a first method for inhibiting the onset ofan osteopontin-related disorder in a subject comprising administering tothe subject a prophylactically effective amount of a nucleic acid whichspecifically reduces levels of osteopontin.

In addition, this invention provides a first method for treating asubject afflicted with an osteopontin-related disorder in a subjectcomprising administering to the subject a therapeutically effectiveamount of a nucleic acid which specifically inhibits the expression ofosteopontin in the subject's osteopontin-expressing cells.

This invention further provides a second method for inhibiting the onsetof an osteopontin-related disorder in a subject comprising administeringto the subject a prophylactically effective amount of ananti-osteopontin antibody or antigen-binding portion thereof.

This invention further provides a second method for treating a subjectafflicted with an osteopontin-mediated disorder comprising administeringto the subject a therapeutically effective amount of an anti-osteopontinantibody or antigen-binding portion thereof.

This invention further provides two compositions. The first compositioncomprises a nucleic acid which specifically inhibits the expression ofosteopontin in an osteopontin-expressing cell and a pharmaceuticallyacceptable carrier. The second composition comprises an anti-osteopontinantibody or antigen-binding portion thereof and a pharmaceuticallyacceptable carrier.

This invention further provides a first method for determining theamount of osteopontin in a sample comprising (a) contacting the samplewith an anti-osteopontin antibody or antigen-binding portion thereofunder suitable conditions, (b) determining the amount of antibody orantigen-binding portion thereof bound to the sample, and (c) comparingthe amount so determined to a known standard, thereby determining theamount of osteopontin in the sample.

This invention further provides a second method for determining theamount of osteopontin in a sample comprising (a) contacting the sampleunder suitable conditions with a nucleic acid which specificallyhybridizes to osteopontin-encoding mRNA, (b) determining the amount ofnucleic acid so hybridized, (c) comparing the amount of nucleic acid sodetermined to a known standard so as to determine the amount ofosteopontin-encoding mRNA in the sample, and (d) comparing the amount ofmRNA so determined to a known standard, thereby determining the amountof osteopontin in the sample.

This invention provides a kit for practicing the first and secondquantitative methods comprising (a) an agent selected from the groupconsisting of (i) an anti-osteopontin antibody or antigen-bindingportion thereof and (ii) a nucleic acid which specifically hybridizeswith osteopontin-encoding mRNA, and (b) instructions for use.

This invention also provides two methods (“assays”) for determiningwhether an agent reduces the amount of osteopontin in anosteopontin-expressing cell. The first assay comprises (a) contactingthe cell with the agent under suitable conditions, (b) determining theamount of osteopontin in the cell, and (c) comparing the amount sodetermined to the amount of osteopontin in a comparable cell in theabsence of the agent, thereby determining whether the agent reduces theamount of osteopontin in the cell.

The second assay comprises the steps of (a) contacting the agent withosteopontin under suitable conditions, (b) determining the activity ofosteopontin in the presence of the agent, and (c) comparing the activityso determined to the activity of osteopontin in the absence of theagent, thereby determining whether the agent reduces the activity ofosteopontin.

This invention further provides a method of treating a subject afflictedwith multiple sclerosis comprising administering to the subject atherapeutically effective amount of an expressible nucleic acid encodingosteopontin. Also provided is a method of inhibiting the onset ofmultiple sclerosis in a subject comprising administering to the subjecta prophylactically effective amount of an expressible nucleic acidencoding osteopontin.

This invention further provides kits for treating or preventing anosteopontin-related disorder. The first kit comprises a nucleic acidwhich specifically inhibits the expression of osteopontin in anosteopontin-expressing cell, and instructions for using the nucleic acidin the treatment or prophylaxis of an osteopontin-related disorder. Thesecond kit comprises an anti-osteopontin antibody or antigen-bindingportion thereof, and instructions for using the antibody orantigen-binding portion thereof in the treatment or prophylaxis of anosteopontin-related disorder.

Finally, this invention provides two methods for treating a subjectafflicted with a disorder mediated by an endogenous protein. The firstmethod comprises administering to the subject (a) osteopontin and (b)the endogenous protein or an antigenic portion thereof, wherein theosteopontin and endogenous protein or antigenic portion thereof areadministered in amounts effective to treat the subject. The secondmethod for treating a subject afflicted with a disorder mediated by anendogenous protein comprises administering to the subject (a) anexpressible osteopontin-encoding nucleic acid and (b) an expressiblenucleic acid encoding the endogenous protein or an antigenic portionthereof, wherein the nucleic acids are administered in amounts effectiveto treat the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A Frequency distribution. Clones with a count higher than 6 wereorganized in decreasing order according to their frequency. Arrowsindicate the three most common ESTs in each library. MBP was the mosthighly expressed gene in all three libraries. GFAP and PLP were the nextmost abundant species in the MS libraries, but their frequency order wasreversed in the control library. Unidentified ESTs are shown in lightercolor than known, annotated clones.

FIG. 1B Category distribution. Clones were distributed into one of thefollowing categories: RN, redundant novel; RK, redundant known; HA, highabundance; SN, solitary novel; and SK, solitary known. The relativecontribution to each category is shown in a pie chart for all libraries.

FIG. 1C Intersectional queries. All possible comparisons were performedamong the three libraries. Clones were counted and distributed intotheir corresponding intersection on the Venn diagrams. The total numberof sequenced clones is shown for each library. The number of differentmRNA species for each library is also shown along with the number ofunknown genes in parentheses. The number of RNA species that werespecific for each library or intersection of libraries is displayedunderlined, along with the number of unknown genes in parentheses.

FIGS. 2A and 2B OPN in macrophages is shown in the center of an activelydemyelinating MS plaque (1A) and in white matter astrocytes adjacent toan active MS plaque (1B). OPN staining was performed with a polyclonalanti-OPN antibody on paraffin-embedded sections. All photos in FIGS.2A-2H are immunoperoxidase-stained with diaminobenzidine chromogen andhematoxylin counterstain. Magnifications: A-D, F, H=370×; E, G=494×.

FIGS. 2C-2F These Figures show relapsing-remitting EAE in mice. EAE wasinduced in nine SJL mice (The Jackson Laboratory, Bar Harbor, Me.) withPLP 139-151, as previously described (24). Four mice injected with PBSserved as controls. Immunostaining was performed with the anti-OPNantibody MPIIIB10₁ (Developmental Studies Hybridoma bank, Iowa City,Iowa) (25) and slides were examined by a blinded observer. In FIG. 2C,OPN was broadly expressed in CNS microglia, especially near inflammatorylesions, but not in adjacent peripheral nerves (arrow). In FIG. 2D,expression in neurons (arrows) was detectable during the acute phase(N=4) and relapse (N=2), but not during remission (N=1) or in mice withinflammatory lesions which never developed paralysis (N=2). OPNexpression in astrocytes (arrows) (FIG. 2E) and choroid plexus cells(FIG. 2F) was also more frequent and more pronounced in immunized mice(100%) than in controls (25%).

FIG. 2G This Figure relates to acute EAE in rat. EAE was induced in 19Lewis rats (The Jackson Laboratory) as described in (13) but with 400 μgGPSCH. Four rats injected with CFA alone served as controls. Brains wereprocessed and stained with MPIIIB10₁. Microglial expression of OPNaround inflammatory lesions (G) correlated with the clinical diseaseseverity. OPN was also expressed in neurons (arrows), mostly in theanimals with severe clinical signs.

FIG. 2H This Figure shows a positive control, i.e., OPN staining in thebony growth plate of a mouse femur with MPIIIB10₁.

FIGS. 3A-3C These Figures relate to clinical attenuation of EAE inOPN−/− mice. OPN+/+ and OPN−/− mice are 129/C57BL/6 mixed background,maintained as a partially outbred strain (30). EAE was induced in OPN+/+(N=18) (closed circles) and OPN−/− (N=17) (open circles) mice (30) withMOG 35-55, as described (31). EAE was scored as follows: 0=normal;1=monoparesis; 2=paraparesis; 3=paraplegia; 4=Tetraparesis; and5=moribund or dead. For each animal, a remission was defined by adecrease of the score of at least one point for at least two consecutivedays. EAE was considered remitting when at least one remission occurredwithin the first 26 days, and progressive when no remission occurred.

In FIG. 3A, OPN−/− (open circles) mice have milder disease than OPN+/+controls (closed circles). The error bars represent the standard errorfor each point. EAE was observed in 100% of both OPN+/+ and OPN−/− micewith MOG 35-55, [N=18 for OPN+/+ and N=17 for OPN−/−]. Although EAEcould be induced with a 100% incidence in OPN−/− mice, a significantlyreduced severity of disease developed in the OPN−/− group, with adecrease of the mean EAE score (at day 30, mean EAE score 2.5 in OPN+/+,compared with 1.2 in OPN−/−, Mann-Whitney Rank Sum test p=0.0373) and adecrease of the mean maximum severity score (mean severity 3.7 inOPN+/+, compared to 2.8 in OPN−/−, Mann-Whitney Rank Sum test p=0.0422).There was no significant delay of the day of disease onset (mean 11.7days in OPN+/+, compared to 12.5 days in OPN−/−, Mann-Whitney Rank Sumtest p=0.322).

In FIG. 3B, OPN−/− mice (open circles) are protected from EAE-relateddeath, with no mice dead out of 17, as compared with 7 dead out of 18among the OPN+/+ mice at day 70 (p=0.0076 by Fisher's exact test).

FIG. 3C shows that OPN promotes progressive EAE. The bars represent thepercentage of mice having a remitting (black) or progressive (white)disease in each group. OPN−/− mice showed a distinct evolution of EAE,with a much higher percentage of mice having remissions compared to thecontrols. (10 out of 18 had remissions in the OPN+/+ group (55.5%),compared to 16 out of 17 in the OPN−/− group (94.1%), p=0.0178 byFisher's exact test).

FIG. 4A This Figure shows inhibition of T cell proliferation in OPN−/−mice. A proliferation assay was performed on draining lymph nodes (LN)from OPN+/+ (closed circles) and OPN−/− (open circles) mice (30), 14days after induction of EAE. EAE was induced with MOG 35-55, asdescribed (31). Draining LN were removed 14 days after immunization, andLN cells were stimulated in 96-well flat bottom plates (2.5 10⁶/ml, 200ml/well) with serial dilutions of HPLC-purified MOG 35-55 (0-50 mM), asdescribed (39). The medium contained 2% serum from the type of mousetested, in order to avoid introducing OPN into the in vitro assays onOPN−/− cells. OPN+/+ normal mouse serum was used for the assays onOPN+/+ cells, while OPN−/− normal mouse serum was used for the assays onOPN−/− cells. Concanvalin A (2 mg/ml), a non-specific mitogen for Tcells, was used as a non-specific positive control. [³H ] thymidine wasadded to the triplicates [mean±standard deviation graphed], after 72 hof antigen stimulation and its incorporation by the proliferating cells(in cpm) was measured 24 h later.

FIG. 4B This Figure shows that OPN−/− cells produce more IL-10 thanOPN+/+ cells. OPN+/+ (black bars) and OPN−/− (white bars) LN cells werestimulated in the same way as for the proliferation assay (FIG. 4A), butin 24-well flat bottom plates (2 ml/well). MOG 35-55 was used at aconcentration of 12.5 mM. IL-10 was measured by ELISA on the 48 hsupernatants (dilution 1/2), in duplicate [mean±standard deviationgraphed], according to the manufacturer's instructions (OPTEIA kit,PharMingen, San Diego, Calif.).

FIG. 4C This Figure shows that OPN−/− cells produce less IFN-gamma thanOPN+/+ cells. OPN+/+ (filled bars) and OPN−/− (open bars) spleen cellswere removed 14 days after induction of EAE with MOG 35-55 andstimulated as described in FIG. 4B, but with 4.5 10⁶ cells/well.IFN-gamma was measured by ELISA on the 48 h supernatants (dilution 1/5),in triplicate, according to the manufacturer's instructions (OPTEIA kit,PharMingen, San Diego, Calif.).

FIG. 4D This Figure shows that OPN−/− cells produce less IL-12 thanOPN+/+ cells. OPN+/+ (black bars) and OPN−/− (white bars) spleen cellswere removed as described for FIG. 4C. IL-12 p70 was measured by ELISAon the 24 h supernatants (dilution 1/1), in duplicate [mean±standarddeviation graphed], according to the manufacturer's instructions (OPTEIAkit, PharMingen, San Diego, Calif.).

FIG. 5 This Figure shows that treatment with DNA encoding osteopontinreduces the incidence and severity of EAE. C57B6 mice were treated withDNA encoding osteopontin.

FIG. 6 This Figure shows that treatment with DNA encoding osteopontinreduces the incidence and severity of EAE. C57B6 mice were treated withDNA encoding osteopontin.

FIG. 7 This Figures shows antibody titers in the mice shown in FIG. 6.

DETAILED DESCRIPTION

Definitions

“Activity” of osteopontin shall mean any enzymatic or binding functionperformed by that protein. Osteopontin activity includes, for example,binding to CD44.

“Antibody” shall include, by way of example, both naturally occurringand non-naturally occurring antibodies. Specifically, this term includespolyclonal and monoclonal antibodies, and fragments thereof.Furthermore, this term includes chimeric antibodies and wholly syntheticantibodies, and fragments thereof.

“Anti-sense nucleic acid” shall mean any nucleic acid which, whenintroduced into a cell, specifically hybridizes to at least a portion ofan mRNA in the cell encoding a protein (“target protein”) whoseexpression is to be inhibited, and thereby inhibits the target protein'sexpression.

“Catalytic nucleic acid” shall mean a nucleic acid that specificallyrecognizes a distinct substrate and catalyzes the chemical modificationof this substrate.

“Comparable cell” shall mean a cell whose type is identical to that ofanother cell to which it is compared. Examples of comparable cells arecells from the same cell line.

“DNAzyme” shall mean a catalytic nucleic acid that is DNA or whosecatalytic component is DNA, and which specifically recognizes andcleaves a distinct target nucleic acid sequence, which can be either DNAor RNA. Each DNAzyme has a catalytic component (also referred to as a“catalytic domain”) and a target sequence-binding component consistingof two binding domains, one on either side of the catalytic domain.

“Endogenous protein” shall mean, with respect to a particular subject, aprotein originally encoded by the subject's own genome.

“Expressible nucleic acid” shall mean a nucleic acid encoding a nucleicacid of interest and/or a protein of interest, which nucleic acid is anexpression vector, plasmid or other construct which, when placed in acell, permits the expression of the nucleic acid or protein of interest.Expression vectors and plasmids are well known in the art.

“Inhibiting” the onset of a disorder shall mean either lessening thelikelihood of the disorder's onset, or preventing the onset of thedisorder entirely. In the preferred embodiment, inhibiting the onset ofa disorder means preventing its onset entirely.

“Inhibiting” the expression of a gene in a cell shall mean eitherlessening the degree to which the gene is expressed, or preventing suchexpression entirely.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

“Osteopontin” shall mean the human protein encoded by the mRNA sequenceset forth in GenBank Accession No. J04765, all naturally occurringvariants and homologues thereof, and where applicable herein, allantigenic fragments thereof.

Active fragments of osteopontin share a functional or binding propertywith full length osteopontin.

Epitopic fragments of osteopontin bind to a monoclonal antibody thatbinds to full length osteopontin.

“Osteopontin-related disorder” shall mean any disorder (a) characterizedby the over-expression of osteopontin in an afflicted subject, (b)ameliorated by inhibiting osteopontin expression in an afflictedsubject, and/or (c) ameliorated by inhibiting osteopontin activity in anafflicted subject, (d) in which expression of osteopontin contributes tothe pathogenesis

Expression of osteopontin that is normal in some individuals maynevertheless contribute toward an osteopontin-related disorder in otherindividuals if such other individuals the osteopontin acts incombinations with another cellular component, such as a protein, inpathogenesis. Some osteopontin-related disorders are characterized by anelevated Th1 immune response and a depressed Th2 immune responserelative to the mean of such responses in a population of normalindividuals (i.e., free of an osteopontin-related disease and not atrisk of such a disease).

Over-expression of osteopontin means an expression level that is greaterthan the mean plus one standard deviation of that in a population ofnormal individuals. Preferably the expression level is at least tentimes the mean of that in a population of normal individuals.

“Ribozyme” shall mean a catalytic nucleic acid molecule which is RNA orwhose catalytic component is RNA, and which specifically recognizes andcleaves a distinct target nucleic acid sequence, which can be either DNAor RNA. Each ribozyme has a catalytic component (also referred to as a“catalytic domain”) and a target sequence-binding component consistingof two binding domains, one on either side of the catalytic domain.

“Specifically hybridize” to a nucleic acid shall mean, with respect to afirst nucleic acid, that the first nucleic acid hybridizes to a secondnucleic acid with greater affinity than to any other nucleic acid.

“Specifically inhibit” the expression of a protein shall mean to inhibitthat protein's expression (a) more than the expression of any otherprotein, or (b) more than the expression of all but 10 or fewer otherproteins.

“Subject” or “patient” shall mean any animal, such as a human, non-humanprimate, mouse, rat, guinea pig or rabbit.

“Suitable conditions” shall have a meaning dependent on the context inwhich this term is used. That is, when used in connection with anantibody, the term shall mean conditions that permit an antibody to bindto its corresponding antigen. When this term is used in connection withnucleic acid hybridization, the term shall mean conditions that permit anucleic acid of at least 15 nucleotides in length to hybridize to anucleic acid having a sequence complementary thereto. When used inconnection with contacting an agent to a cell, this term shall meanconditions that permit an agent capable of doing so to enter a cell andperform its intended function. In one embodiment, the term “suitableconditions” as used herein means physiological conditions.

“Treating” a disorder shall mean slowing, stopping or reversing thedisorder's progression. In the preferred embodiment, treating a disordermeans reversing the disorder's progression, ideally to the point ofeliminating the disorder itself. As used herein, ameliorating a disorderand treating a disorder are equivalent.

The term “immune” response is the development of a beneficial humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against osteopontinan amyloid peptide in a recipient patient. Such a response can be anactive response induced by An “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

Unless otherwise apparent from the context, all elements, steps orfeatures of the invention can be used in any combination with otherelements, steps or features.

General

This invention provides a method for reducing the amount of osteopontinin an osteopontin-expressing cell comprising introducing into the cell anucleic acid which specifically inhibits osteopontin expression in thecell. In one embodiment, this method further reduces the amount ofosteopontin secreted by an osteopontin-secreting cell.

In this method, the nucleic acid can be, for example, DNA or RNA. In thepreferred embodiment, the nucleic acid is DNA.

In addition, the nucleic acid can be an anti-sense nucleic acid thathybridizes to osteopontin-encoding mRNA, or a catalytic nucleic acidthat cleaves osteopontin-encoding mRNA. In the preferred embodiment, thenucleic acid is an expressible nucleic acid encoding an anti-sensenucleic acid that hybridizes to osteopontin-encoding mRNA, and/orencoding a catalytic nucleic acid that cleaves osteopontin-encodingmRNA.

Osteoponin expression can also be inhibited using zinc finger proteinsor nucleic acids encoding the same as described in WO 00/00409.Alternatively, inhibition of expression can be achieved using siRNAs asdescribed by WO 99/32619, Elbashir, EMBO J. 20, 6877-6888 (2001) andNykanen et al., Cell 107, 309-321 (2001); WO 01/29058.

In these methods, the osteopontin-expressing cell can be, for-example, aneuron, a macrophage, a vascular endothelial cell, an astrocyte or amicroglial cell. In the preferred embodiment, the cell is a neuron.

This invention also provides a first method for inhibiting the onset ofan osteopontin-related disorder in a subject comprising administering tothe subject a prophylactically effective amount of a nucleic acid whichspecifically inhibits the expression of osteopontin in the subject'sosteopontin-expressing cells.

In addition, this invention provides a first method for treating asubject afflicted with an osteopontin-related disorder in a subjectcomprising administering to the subject a therapeutically effectiveamount of a nucleic acid which specifically inhibits the expression ofosteopontin in the subject's osteopontin-expressing cells.

In these first methods of prophylaxis and treatment, the nucleic acidcan be, for example, DNA or RNA. In the preferred embodiment, thenucleic acid is DNA.

In addition, the nucleic acid can be an anti-sense nucleic acid thathybridizes to osteopontin-encoding mRNA, or a catalytic nucleic acidthat cleaves osteopontin-encoding mRNA. In the preferred embodiment, thenucleic acid is an expressible nucleic acid encoding an anti-sensenucleic acid that hybridizes to osteopontin-encoding mRNA, and/orencoding a catalytic nucleic acid that cleaves osteopontin-encodingmRNA.

Also in these first methods of prophylaxis and treatment, the subject'scells in which the amount of osteopontin is reduced can be, for example,neurons, macrophages, vascular endothelial cells, astrocytes ormicroglial cells.

This invention further provides a second method for inhibiting the onsetof an osteopontin-related disorder in a subject comprising administeringto the subject a prophylactically effective amount of ananti-osteopontin antibody or antigen-binding portion thereof.

This invention further provides a second method for treating a subjectafflicted with an osteopontin-mediated disorder comprising administeringto the subject a therapeutically effective amount of an anti-osteopontinantibody or antigen-binding portion thereof.

In the preferred embodiment of the first and second methods ofprophylaxis and treatment, the osteopontin-related disorder is multiplesclerosis. Preferably, the subject is a human.

This invention further provides two compositions. The first compositioncomprises a nucleic acid which specifically inhibits the expression ofosteopontin in an osteopontin-expressing cell and a pharmaceuticallyacceptable carrier. The second composition comprising ananti-osteopontin antibody or antigen-binding portion thereof and apharmaceutically acceptable carrier.

Determining a therapeutically or prophylactically effective amount ofthe instant compositions can be done based on animal data using routinecomputational methods. In one embodiment, the therapeutically orprophylactically effective amount contains between about 0.1 mg andabout 1 g of nucleic acid or protein, as applicable. In anotherembodiment, the effective amount contains between about 1 mg and about100 mg of nucleic acid or protein, as applicable. In a furtherembodiment, the effective amount contains between about 10 mg and about50 mg of the nucleic acid or protein, as applicable.

In this invention, administering the instant compositions can beeffected or performed using any of the various methods and deliverysystems known to those skilled in the art. The administering can beperformed, for example, intravenously, orally, via implant,transmucosally, transdermally, intramuscularly, intrathecally, andsubcutaneously. The following delivery systems, which employ a number ofroutinely used pharmaceutical carriers, are only representative of themany embodiments envisioned for administering the instant compositions.

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprylactone. Osteopontin or nucleic acids of the invention canalso be administered attached to particles using a gene gun.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systemsinclude vehicles such as suspending agents (e.g., gums, zanthans,cellulosics and sugars), humectants (e.g., sorbitol), solubilizers(e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g.,sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservativesand antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid),anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

This invention further provides a first method for determining theamount of osteopontin in a sample comprising (a) contacting the samplewith an anti-osteopontin antibody or antigen-binding portion thereofunder suitable conditions, (b) determining the amount of antibody orantigen-binding portion thereof bound to the sample, and (c) comparingthe amount so determined to a known standard, thereby determining theamount of osteopontin in the sample.

In one embodiment of this first quantitative method, the agent is ananti-osteopontin antibody or antigen-binding portion thereof.Preferably, the antibody or antigen-binding portion thereof is labeledwith a detectable marker.

This invention further provides a second method for determining theamount of osteopontin in a sample comprising (a) contacting the sampleunder suitable conditions with a nucleic acid which specificallyhybridizes to osteopontin-encoding mRNA, (b) determining the amount ofnucleic acid so hybridized, (c) comparing the amount of nucleic acid sodetermined to a known standard so as to determine the amount ofosteopontin-encoding mRNA in the sample, and (d) comparing the amount ofmRNA so determined to a known standard, thereby determining the amountof osteopontin in the sample.

In one embodiment of this second quantitative method, the nucleic acidis labeled with a detectable marker.

In the first and second quantitative methods, the sample can be anysample containing or suspected of containing osteopontin alone, or inthe presence of osteopontin-producing cells. In one embodiment, thesample is a tissue sample. Tissue samples include, without limitation,bodily fluid samples such as cerebrospinal fluid and blood and itscomponent parts, and sections of solid tissue such as brain and spinalcord. Tissue samples can comprise, for example, neurons, macrophages,vascular endothelial cells, astrocytes or microglial cells. In oneembodiment, the tissue sample is from a subject afflicted with orsuspected of being afflicted with an osteopontin-related disorder,preferably multiple sclerosis.

In a further embodiment, the first and second quantitative methodscomprise the step of determining the location of osteopontin within thetissue sample. Steps (a) and (b) of these methods can be performed, forexample, either in vivo or ex vivo.

This invention provides a kit for practicing the first and secondquantitative methods comprising (a) an agent selected from the groupconsisting of (i) an anti-osteopontin antibody or antigen-bindingportion thereof and (ii) a nucleic acid which specifically hybridizeswith osteopontin-encoding mRNA, and (b) instructions for use.

This invention also provides two methods (“assays”) for determiningwhether an agent reduces osteopontin activity or the amount ofosteopontin in an osteopontin-expressing cell. The first assay comprises(a) contacting the cell with the agent under suitable conditions, (b)determining the amount of osteopontin in the cell, and (c) comparing theamount so determined to the amount of osteopontin in a comparable cellin the absence of the agent, thereby determining whether the agentreduces the amount of osteopontin in the cell.

In the first assay, the osteopontin-expressing cell can be, for example,a neuron, a macrophage, a vascular endothelial cell, an astrocyte and amicroglial cell. Preferably, the cell is a human cell. In step (b) ofthe first assay, the amount of osteopontin in the cell is determinedusing an anti-osteopontin antibody or antigen-binding portion thereof.Alternatively, in step (b), the amount of osteopontin in the cell isdetermined using a nucleic acid which specifically hybridizes withosteopontin-encoding mRNA.

The second assay comprises the steps of (a) contacting the agent withosteopontin under suitable conditions, (b) determining the activity ofosteopontin in the presence of the agent, and (c) comparing the activityso determined to the activity of osteopontin in the absence of theagent, thereby determining whether the agent reduces the activity ofosteopontin.

In one embodiment of the second assay, the osteopontin is in a cell.This cell can be, for example, a neuron, a macrophage, a vascularendothelial cell, an astrocyte and a microglial cell. Preferably, thecell is a human cell.

This invention further provides methods for treating a subject afflictedwith a disorder mediated by an endogenous protein. One method comprisesadministering to the subject (a) osteopontin and (b) the endogenousprotein or an antigenic portion thereof, wherein the osteopontin andendogenous protein or antigenic portion thereof are administered inamounts effective to treat the subject.

In this method, the osteopontin and endogenous protein or antigenicportion thereof can be administered simultaneously. Alternatively, theosteopontin and endogenous protein or antigenic portion thereof areadministered separately.

A second method for treating a subject afflicted with a disordermediated by an endogenous protein comprises administering to the subject(a) an expressible osteopontin-encoding nucleic acid and (b) anexpressible nucleic acid encoding the endogenous protein or an antigenicportion thereof, wherein the nucleic acids are administered in amountseffective to treat the subject.

In this second method, the osteopontin-encoding nucleic acid and nucleicacid encoding the endogenous protein or antigenic portion thereof can beadministered simultaneously, either on the same vector or separatevectors. Alternatively, the osteopontin-encoding nucleic acid andnucleic acid encoding the endogenous protein or antigenic portionthereof are administered separately.

In one embodiment of the first and second methods for treating a subjectafflicted with a disorder mediated by an endogenous protein, thedisorder is an autoimmune disorder. In another embodiment, the disorderis multiple sclerosis, insulin-dependent diabetes mellitus, rheumatoidarthritis, autoimmune uveitis, primary billiary cirrhosis or Alzheimer'sdisease. Preferably, the disorder is multiple sclerosis.

In the first and second methods where the disorder treated is multiplesclerosis, the endogenous protein can be, for example, myelin basicprotein, proteolipid protein, myelin-associated glycoprotein, cyclicnucleotide phosphodiesterase, myelin-associated oligodendrocytic basicprotein, or alpha-B-crystalin.

Table 1 sets forth examples of disorders treatable by the instantmethods, and their corresponding endogenous proteins.

TABLE 1 Human Endogenous Protein-Mediated Disorders AutoimmuneSelf-Protein(s) Associated With An Disease Tissue Targeted AutoimmuneDisease Multiple sclerosis central nervous myelin basic protein,proteolipid protein, system myelin associated glycoprotein, cyclicnucleotide phosphodiesterase, yelin- associated glycoprotein, myelin-associated oligodendrocytic basic protein; alpha-B-crystalin GuillianBarre peripheral nerv. peripheral myelin protein I and others Syndromesys. Insulin Dependent □ cells in islets of tyrosine phosphatase IA2,IA-2b; Diabetes Mellitus pancreas glutamic acid decarboxylase (65 and 67kDa forms), carboxypeptidase H, insulin, proinsulin, heat shockproteins, glima 38, islet cell antigen 69 KDa, p52, gangliosideantigens, islet cell glucose transporter GLUT-2 Rheumatoid synovialjoints Immunoglobulin, fibrin, filaggrin, type Arthritis I, II, III, IV,V, IX, and XI collagens, GP-39, hnRNPs Autoimmune Uveitis eye, uveaS-antigen, interphotoreceptor retinoid binding protein (IRBP),rhodopsin, recoverin Primary Biliary biliary tree of pyruvatedehydrogenase complexes (2- Cirrhosis liver oxoacid dehydrogenase)Autoimmune Liver Hepatocyte antigens, cytochrome P450 HepatitisPemphigus vulgaris Skin Desmoglein-1, −3, and others Myasthenia Gravisnerve-muscle acetylcholine receptor junct. Autoimmune stomach/parietalH⁺/K⁺ ATPase, intrinsic factor gastritis cells Pernicious Anemia Stomachintrinsic factor Polymyositis Muscle histidyl tRNA synthetase, othersynthetases, other nuclear antigens Autoimmune Thyroid Thyroglobulin,thyroid peroxidase Thyroiditis Graves's Disease ThyroidThyroid-stimulating hormone receptor Vitiligo Skin Tyrosinase,tyrosinase-related protein-2 Systemic Lupus Systemic nuclear antigens:DNA, histones, Eryth. ribonucleoproteins Celiac Disease Small bowelTransglutaminase Pathologic Neurodegenerative Disease DeformityEndogenous Protein Alzherimer's disease senile plaques amyloid □ proteinParkinson's disease Lewy bodies □-synuclein Huntington's diseaseintranuclear Huntingtin protein inclusions Prion disease Prion proteinPrion protein inclusions Endogenous Proteins Disease AbnormalityAssociated With Disease Obesity weight gain due to syndecan-3,perilipin, Orexin, energy intake > expenditure Galanin, glucogon-likepeptide receptor, Osteoarthritis cartilage degeneration cathepsins,plasmin, collagenases, metalloproteinases Spinal cord injury inhibitionof Nogo-1 regeneration Hypertension persistent high bloodangiotensin-converting enzyme pressure Peptic ulcer disease excessstomach acid H⁺/K⁺ ATPase, gastrin Aging superoxide dismutase Depressionexcessive serotonin serotonin 5HT2 receptor, □₁- adrenergic receptorGout Excess uric acid Xanthine oxidase Migraine headaches vasospasmserotonin 5HT_(1B) and 5HT_(1D) receptors Hyperlipidemia elevated lipidsHMG CoA-reductase, apolipoproteins A, B-100 Coronary artery obstructionof Angiotensin-converting enzyme, disease coronary arteriesapolipoproteins A, B-100 restricting blood flow

Finally, this invention provides kits for treating or preventing anosteopontin-related disorder. The first kit comprises a nucleic acidwhich specifically inhibits the expression of osteopontin in anosteopontin-expressing cell, and instructions for using the nucleic acidin the treatment or prophylaxis of an osteopontin-related disorder. Thesecond kit comprises an anti-osteopontin antibody or antigen-bindingportion thereof, and instructions for using the antibody orantigen-binding portion thereof in the treatment or prophylaxis of anosteopontin-related disorder.

Methods of Treatment Using Osteoponin or Nucleic Acids EncodingOsteononin

The invention provides methods of treating osteopontin related disordersusing osteopontin, epitopic fragments thereof or nucleic acids encodingeither of these, graft versus host disease. These methods are useful fortreating a variety of disease for which downregulation of type 1 immuneresponse and or upregulation of type 2 immune response is required. Suchdiseases include autoimmune diseases, host versus graft disease andgraft versus host disease, granulomatous disorder, herpes simplexkeratisits, bacterial arthritis and epilepsy. Autoimmune diseasesinclude multiple sclerosis, rheumatoid arthritis, and type I diabetes.The methods can be used to treat or prevent such disorders in patientshaving or at risk of such disorders. Patients having a disorder includethose who are currently experiencing clinical symptoms, and patients whoexperience symptoms intermittently who may be symptomatic orasymptomatic at any particular time. These methods are particularlyeffective for treating a subject afflicted with multiple sclerosiscomprising administering to the subject a therapeutically effectiveamount of an expressible nucleic acid encoding osteopontin. Alsoprovided is a method of inhibiting the onset of multiple sclerosis in asubject comprising administering to the subject a prophylacticallyeffective amount of an expressible nucleic acid encoding osteopontin.

1. Nucleic Acids Encoding Osteopontin

The nucleic acids used in these methods encodes osteopontin or anepitopic fragment thereof. The nucleic acids are transcribed andtranslated (DNA) or transcribed (mRNA) in situ, and the translationproduct generates an immune response. DNA immunization is described byWO 99/28471, Chowdhury et al., PNAS 95, 669-674 (1998) and J. Immunol.Methods 231, 83-91 (1999)).

DNA immunization can be performed with or without an adjuvant. Theadjuvant, if present, can be one that is typically used with a proteinantigen (see below), or it can be an adjuvant that is specificallychosen to associate with DNA, such as the positively charged detergentCTAB. The DNA can be administered naked or complexed with colloidalmaterials. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,4,946,787; and 4,897,355) and lipofection reagents are sold commercially(e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids thatare suitable for efficient lipofection of polynucleotides include thoseof Felgner, WO 91/17424, WO 91/16024.

Optionally, the DNA is dissolved in a pharmaceutical carrier in asolution that is sterile and substantially isotonic.

The nucleic acid used as an immunogen contains a segment encodingosteopontin and other segments encoding one or more regulatory sequencesthat ensure translation and transcription (in the case of DNA) of theimmunogen. Regulatory sequences include a promoter, enhancer,transcription termination site, ribosome binding site, and intronicsites. The promoter can be constitutive or inducible or tissue specific,in which case the promoter is preferably specific for antigen presentingcells. For expression in blood cells, as is desirable for induction ofan immune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. Optionally, DNAimmunogens is present as a component of a vector. In some instances, thevector encodes proinflammatory cytokines to attract immune cells to thesite of injection. In some instances, the DNA encodes a fusion protein,comprising an antigenic component to which antibodies are desired and aT-cell antigen, such as tetanus toxoid, or other adjuvant such as C3d(see Dempsey et al., Science 271, 348-50 (1996)). The DNA can encode afull length protein or a desired epitopic fragment thereof.

In a further variation, the nucleic acid can be incorporated into thegenome of a virus or a bacteria. Optionally, the nucleic acid isincorporated in such a manner that the immunogenic peptide is expressedas a secreted protein or as a fusion protein with an outer surfaceprotein of a virus or a transmembrane protein of a bacteria so that thepeptide is displayed. Viruses or bacteria used in such methods should benonpathogenic or attenuated. Suitable viruses include adenovirus, HSV,Venezuelan equine encephalitis virus and other alpha viruses, vesicularstomatitis virus, and other rhabdo viruses, vaccinia and fowl pox.Suitable bacteria include Salmonella and Shigella. Fusion of animmunogenic peptide to HBsAg of HBV is particularly suitable.

2. Administration of Osteopontin

The invention also provides methods in which osteopontin or an epitopicfragment thereof is administered to a patient. The osteopontin generatesan immune response which lowers levels of osteopontin in the patient inthe same manner as described for nucleic acids encoding osteopontinadministration. Osteopontin can be administered alone or fused as acomponent of a longer protein. Optionally such a fusion protein caninclude a heterologous amino acid sequence that induces a helper T-cellresponse against the heterologous amino acid sequence and thereby aB-cell response against osteopontin.

Epitopic fragments of osteopontin suitable for use in the methods can beinitially screened by standard computer programs that identify regionsof probable immunogenicity. Fragments are then tested for activity inanimal models as described in the Examples.

3. Adjuvants

A variety of adjuvants can be used in combination with osteopontin orepitopic fragments thereof or nucleic acids encoding the same to elicitan immune response. Preferred adjuvants include aluminum hydroxide andaluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) (seeGB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Mont., now part ofCorixa). Stimulon™ QS-21 is a triterpene glycoside or saponin isolatedfrom the bark of the Quillaja Saponaria Molina tree found in SouthAmerica (see Kensil et al., in Vaccine Design: The Subunit and AdjuvantApproach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.5,057,540),(Aquila BioPharmaceuticals, Framingham, Mass.). Otheradjuvants are oil in water emulsions (such as squalene or peanut oil),optionally in combination with immune stimulants, such as monophosphoryllipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Anotheradjuvant is CpG (WO 98/40100).

4. Therapeutic and Prophylactic Regimes for Generating an ImmuneResponse

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,an osteoponin related disorder in an amount sufficient to eliminate orreduce the risk, lessen the severity, or delay the outset of thedisease, including biochemical, histologic and/or behavioral symptoms ofthe disease, its complications and intermediate pathological phenotypespresenting during development of the disease. In therapeuticapplications, compositions or medicants are administered to a patientsuspected of, or already suffering from such a disease in an amountsufficient to cure, or at least partially arrest, the symptoms of thedisease (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. An effective regimecomprises a combination of an effective dosage and frequency ofadministration. Typically, the immune response is monitored and repeateddosages are given if the immune response starts to wane.

Effective doses of the compositions of the present invention varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. The amount of immunogendepends on whether adjuvant is also administered, with higher dosagesbeing required in the absence of adjuvant. The amount of immunogendepends on whether adjuvant is also administered, with higher dosagesbeing required in the absence of adjuvant. The amount of an immunogenfor administration sometimes varies from 1-500 μg per patient and moreusually from 5-500 μg per injection for human administration.Occasionally, a higher dose of 1-2 mg per injection is used. Typicallyabout 10, 20, 50 or 100 μg is used for each human injection. The timingof injections can vary significantly from once a day, to once a year, toonce a decade. A typical regimen consists of an immunization followed bybooster injections at time intervals, such as 6 week intervals. Anotherregimen consists of an immunization followed by booster injections 1, 2and 12 months later. Another regimen entails an injection every twomonths for life. Alternatively, booster injections can be on anirregular basis as indicated by monitoring of immune response.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient.

In order to facilitate an understanding of the Experimental Detailssection that follows, certain frequently occurring methods and/or termsare best described in Sambrook, et al. (40).

This invention will be better understood by reference to the Exampleswhich follow, but those skilled in the art will readily appreciate thatthe information detailed is only illustrative of the invention asdescribed more fully in the claims which follow thereafter.

EXAMPLES

Synopsis

Multiple sclerosis is a demyelinating disease, characterized byinflammation in the brain and spinal cord, possibly due to autoimmunity.Large scale sequencing of cDNA libraries, derived from plaques dissectedfrom brains of patients with MS, indicated an abundance of transcriptsfor osteopontin. Microarray analysis of spinal cords from rats paralyzedfrom experimental autoimmune encephalomyelitis (“EAE”), a model of MS,also revealed increased OPN transcripts. Osteopontin-deficient mice wereresistant to progressive EAE and had frequent remissions, andmyelin-reactive T cells in OPN−/− mice produced more IL-10 and lessγ-interferon than in OPN+/+ mice. Osteopontin thus appears to regulateTh1-mediated demyelinating disease, and offers a target in blockingdevelopment of progressive MS, as well as other osteopontin-mediateddiseases.

Discussion

We investigated a role for OPN in MS and in an experimental model forMS, experimental autoimmune encephalomyelitis (“EAE”) in mice. Initiallywe set out to identify gene transcripts involved in the inflammatoryresponse that might be increased in the central nervous system (“CNS”)during active EAE, and that returned to nomial when EAE was successfullytreated after the onset of paralysis. Customized oligonucleotidemicroarrays were produced to monitor transcription of genes involved ininflammatory responses (12 and 13). Details concerning these custommicroarrays are set forth in the Methods section below. These initialmicroarray experiments showed that osteopontin transcripts were elevatedin the brains of rats with EAE, and not in brains of rats protected fromEAE. Details of these experiments and others relevant to the subjectinvention are set forth in the Methods section below.

In parallel, we performed high throughput sequencing of expressedsequence tags [EST], utilizing non-normalized cDNA brain libraries (15,16, 17), generated from MS brain lesions and control brain. Using thisprotocol the mRNA populations present in the brain specimens areaccurately represented, enabling the quantitative estimation oftranscripts and comparisons between specimens (Table 2 and Table 3).Molecular mining of two sequenced libraries and their comparison with anormal brain library, matched for size and tissue type, and constructedwith an identical protocol, revealed that OPN transcripts werefrequently detected, and were exclusive to the MS mRNA population, butnot found in control brain mRNA (Table 2).

TABLE 2 MS-Specific Gene Transcripts MS1 MS2 Average Genomic Accession #Geno description abundance abundance clone count Cellular functionlocation S45630 Alpha B-crystallin 7 12 9.5 cell structure/motility11q22.3-q23.1 M61901 Prostaglandin D synthase 8 7 7.5 cell signaling/9q34.2-34.3 cell communication X75252 Prostatic binding protein 6 7 6.5cell signaling/ 12q24.1* cell communication X53777 Ribosomal protein L1710 2 6 gene/protein expression 18q X13694 Osteopontin 8 3 5.5 cellstructure/ 4q21-q25 motility/signaling AB037797 KIAA1376 6 3 4.5unclassified 5 Z19554 Vimentin 4 5 4.5 cell structure/motility 10p13X52947 Cardiac gap junction protein 5 3 4 cell signaling/ 6q21-q23.2*cell communication D17554 DNA-binding protein 4 4 4 gene/proteinexpression 12q23-24.1* AF181862 G protein-coupled receptor 2 6 4 cellsignaling/ 16p12 cell communication AB018321 ATPase Na/K transporting, 26 4 cell signaling/ 1q21-q23 alpha 2 (KIAA0778) cell communicationAF100620 MORF-related gene X 1 7 4 gene/protein expression Xq22*AB002363 KIAA0365 1 7 4 unclassified 19p12 AF072902 Gp130 associatedprotein GAM 6 1 3.5 unclassified 19p13.3 M11233 Cathepsin D 6 1 3.5cell/organism defense 11p15.5 D78014 Dihydropyrimidinase relatedprotein-3 6 1 3.5 metabolism 5q32 X53305 Stathmin 4 3 3.5 cell division1p36.1-p35* AF026844 Ribosomal protein L41 4 3 3.5 unclassified 22q12U48437 Human amyloid precursor-like protein 1 4 3 3.5 unclassified N/AU51678 Small acidic protein 3 4 3.5 unclassified N/A U67171Selenoprotein W 3 4 3.5 metabolism 19q13.3* S80794 Tyrosine andtryptophan 2 5 3.5 cell signaling/ 22q12.3 hydroxylase activator cellcommunication AB011089 KIAA0517 (brain) 4 2 3 unclassified 4q28 AAD32952PHR1 isoform 4 [Mus musculus] 3 3 3 unclassified N/A J04173Phosphoglycerate mutase, brain 2 4 3 metabolism 10q25.3 M22382 Heatshock 60 kD protein 1 (chaperonin) 2 4 3 cell/organism defense 2 M34671HUMCD59A Human lymphocytic 2 4 3 unclassified 11p13 antigen CD59/MEM43M64786 Similar to Myc 2 4 3 unclassified N/A AJ132695 Rac1 gene 2 4 3cell signaling/ Xq26.2-27.2 cell communication Z99716 Septin 3 1 5 3cell division 22q13.1 U49436 Human translation initiation factor 5 1 5 3gene/protein expression 14q32* CAA63354 Cysteine string protein [Bostaurus] 2 3 2.5 unclassified N/A U90915 Cytochrome c oxidase subunit IV4 1 2.5 metabolism 16q24.1 J02611 Apolipoprotein D 4 1 2.5 metabolism3q26.2-qter X05607 Cystatin C (cysteine proteinase 4 1 2.5 metabolism20p11.2 inhibitor precursor) U45976 Clathrin assembly protein 4 1 2.5unclassified 11q14 lymphoid myeloid leukemia J00272 Metallothionein-IIpseudogene 4 1 2.5 unclassified 4p11-q21 S69965 Beta-synuclein 3 2 2.5unclassified 5q35 Y00711 Lactate dehydrogenase B 3 2 2.5 metabolism12p12.2-p12.1 L37033 FK-506 binding protein homologue 3 2 2.5 cellsignaling/ 19p12 (FKBP38) cell communication AF044956 NADH: ubiquinoneoxidoreductase 3 2 2.5 metabolism 8q13.3 B22 subunit AB011154 KIAA0582(brain) 3 2 2.5 unclassified 2p12 X55039 Centromere autoantigen B 3 22.5 unclassified 20p13 X64364 Basigin 3 2 2.5 cell signaling/ 19p13.3cell communication U82761 S-adenosyl homocysteine hydrolase-like 1 3 22.5 metabolism 1 D13627 Chaperonin containing TCP1, 2 3 2.5 gene/proteinexpression 21q22.11 subunit B (theta) Z47087 Transcription elongationfactor 2 3 2.5 gene/protein expression 5q31 B (SIII), polypeptide likeX75861 Testis enhaced gene transcript 2 3 2.5 cell division 12q12-q13M16447 Quinoid dihydropteridine reductase 2 3 2.5 metabolism 4p15.31M22918 Non-muscle myosin alkali light chain 2 3 2.5 unclassified 12M55270 Matrix Gla protein 2 3 2.5 unclassified 12p13.1-p12.3 AF151807CGI-49 protein 2 3 2.5 unclassified 1 AAD45960 Human EST H08032.1 (NID:g872854) 2 3 2.5 unclassified 7q11.23-q21.1 Only genes with a mean foldchange of >2.5 are listed. “N/A” indicates that mapping position is notknown. *indicates genomic regions that reached nominal criteria oflinkage in genome-wide screenings.

We sequenced more than 11,000 clones from MS libraries 1 and 2, andcontrol libraries (FIGS. 1A-1C), and focused our analysis on genespresent in both MS libraries, but absent in the control library. Thisyielded 423 genes, including 26 novel genes. From those, 54 genes showeda mean fold change of 2.5 or higher in MS libraries 1 and 2 (Table 2).Transcripts for alpha B-crystallin, an inducible heat shock protein,localized in the myelin sheath, and known to be targeted by T cells inMS, were the most abundant transcripts unique to MS plaques (19) (Table2). The next five most abundant transcripts, included those forprostaglandin D synthase, prostatic binding protein, ribosomal proteinL17, and OPN.

Next, we analyzed all genes present in each of the three cDNA libraries,and found 330 (seven novel) genes. Based on the clone count of eachsequenced gene, a table was constructed with transcripts showing anaverage fold difference equal to or greater than ±2.00 between MS andcontrol. Forty of these transcripts were divided into three levels, onthe basis of the consistency of differential expression across libraries(Table 3). Some of these genes (Table 3) were myelin basic protein(MBP), heat shock protein 70 (HSP-70), glial fibrillary acidic protein(GFAP) and synaptobrevin. MBP transcripts displayed consistent highlevels of expression in the three libraries, indicating a very highturnover rate for this protein. Expression of HSP70-1, which is involvedin myelin folding (20), was significantly elevated. Although notdifferentially expressed, GFAP was among the three most abundant speciesin all the libraries, consistent with a prominent glial (or astrocytic)response in the MS brains. Six genes belonging to the KIAA group oflarge-size cloned mRNAs showed differential expression. The decreasedtranscription of synaptobrevin is important given that it belongs to afamily of small integral membrane proteins specific for synapticvesicles in neurons. Recent evidence indicates that axonal loss is oneof the major components of pathology in MS (21, 22).

TABLE 3 Genes Differentially Expressed in MS and Normal Libraries Clonecount Average Genomic Accession # Gene description MS1 MS2 CTRLFold-difference Cellular function location M17885 Acidic ribosomalphosphoprotein P0 9 13 2 5.2 gene/protein expression 12q24* X16869Elongation factor 1-alpha 32 33 15 2.07 gene/protein expression 6q14M54927 Myelin proteolipid protein 41 16 64 −3 cell structure/motilityXq22* U66623 Small GTPase 1 1 7 −7.35 cell signaling/ 2q21.2 cellcommunication M26252 Piruvate kinase, muscle 2 1 10 −8.04 Energymetabolism 15q22 M59828 Heat shock protein 70-1 2 14 1 7.29cell/organism defense 6q21.3* AF068848 Scaffold attachement factor A 6 11 2.49 Cell division N/A AF035283 Clone 23916 10 15 5 2.36 unclassifiedN/A U46571 Tetratricopeptide repeat protein 2 10 4 3 2.29 unclassified17q11.2 AF131756 clone 24912 2 10 10 −3.02 unclassified N/A X92845 N-mycdownstream regulated 1 7 7 −4 unclassified 8q24.1 M97168 X(Inactive)-specific transcript 1 13 10 −4.33 unclassified Xq13.2AB023167 Neural membrane protein 35 (KIAA0950) 1 3 7 −4.74 unclassified12q13 AB002391 HERC2 (KIAA0393) 2 1 7 −5.63 unclassified 15q13 AF055026RaP2 interacting protein 3 1 1 6 −6.3 Unclassified 17 U89330Microtubute-associated protein 2 1 1 6 −6.3 cell structure/motility2q34-q35 M20020 Ribosomal protein S6 5 6 1 5.23 gene/protein expression9p21* X03747 Na/K-ATPase beta subunit 5 5 1 4.78 Energy metabolism1q22-q25 V00572 Phosphoglycerate kinase 5 4 1 4.33 metabolism Xq13M59488 S100 protein beta-subunit 5 4 1 4.33 cell signaling/ Z1q22.3 cellcommunication AB018271 KIAA0728 (Brain) 4 4 1 3.83 Unclassified6p11-11.2 AB020718 KIAA0911 4 3 1 3.38 unclassified 1p36* AAD02202CsM-KII inhibitory protein 2 5 1 3.26 unclassified N/A [Rattusnorvegicus] D67025 Proteasome 26S subunit 1 1 3 −3.15 cell/organismdefense 17q21.1 (non-ATPase, 3) D63424 Glycogen synthase kinase 3 alpha1 1 3 −3.15 metabolism 19q13.3-13* AF051976 Unconventional myosin XV 1 13 −3.15 cell structure/motility 17p11.2 X13916 LDL-receptor relatedprotein 1 1 3 −3.15 metabolism 12q13.q14 AB028981 KIAA1058 1 1 3 −3.15unclassified 13 AF102846 N-ethylmalelmide-sensitive factor 3 1 5 −3.61metabolism 17q21 L10284 Calnexin 3 1 5 −3.61 cell/organism defense 5q35D88435 Cyclin G associated kinase 1 2 5 −3.85 cell division 4p16AF054987 aldolase C 2 1 5 −4.02 metabolism 17cen-q12 CAB01750 similar toMitochondrial carrier proteins 1 1 4 −4.2 Unclassified N/A[Caenorhabditis elegans] AL137406 Clone DKFZp434M162 1 1 4 −4.2Unclassified N/A AB032436 Brain specific Na+-dependent 1 1 4 −4.2metabolism 19q13* inorganic phosphate cotransporter L10911 Splicingfactor(CC1.3) 1 1 4 −4.2 gene/protein expression 20 L77864 Amyloid beta(A4) precursor 1 4 7 −4.42 unclassified 11p15 protein-binding, family B,member 1 (Fe65) U64520 Synaptobfevin-3 1 1 5 −5.25 unclassified 1p35-p36D87465 KIAA0275 (brain) 1 1 5 −5.25 unclassified 10 Only genescorresponding to transcripts with an average fold difference of ≧2 arelisted. The first section of the table lists genes whose expression wasstatistically significant in both MS libraries when compared to the CTRLlibrary (Fisher's exact test, p < 0.05). The second section containsgenes with significant difference in expression in only one of the MSlibraries and the CTRL. The last section includes genes withnon-significant differences but AFD ≧ ±3.00. N/A, mapping position isnot known. *genomic regions that reached nominal criteria of linkage ingenome-wide screenings.

Given the known inflammatory role for OPN, we examined the cellularexpression pattern of this protein in human MS plaques and in controltissue, by immunohistochemistry. To identify cells expressing OPN insitu we used a polyclonal antibody, generated in mouse againstrecombinant glutathione S-transferase (GST)-OPN, to stain postmortem MSand control tissue samples (23) (FIGS. 2A and 2B). Within active MSplaques OPN was found on microvascular endothelial cells and macrophages(FIG. 2A), and in white matter adjacent to plaques. Reactive astrocytesand microglia also expressed OPN (FIG. 2B).

The role of OPN in inflammatory demyelinating disease was next examinedusing two models of EAE (1). A relapsing-remitting model of EAE wasfirst used to compare the cellular expression of OPN at different stagesof the disease. Disease was induced in SJL mice by immunization with theproteolipid protein peptide 139-151 (PLP139-151) in complete Freund'sadjuvant (CFA), and the animals were scored daily for signs of disease(24). Brain and spinal cord were removed during acute phase, remissionor first relapse. Histopathologic identification of OPN in EAE was thenperformed. (FIGS. 2C-2F). OPN was expressed broadly in microglia duringboth relapse and remission from disease, and this expression was focusednear perivascular inflammatory lesions. In addition to OPN expression onglia, expression in neurons was detectable during acute disease, andrelapse, but not during remission. To confirm the expression of OPN inan acute form of EAE, a rapid, monophasic demyelinating disease wasinduced in Lewis rats (12), then OPN immunostaining was performed ontheir brains (FIG. 2G). OPN expression in microglia and neurons waspredominant in the sick rats, and was focused close to the acutelesions, as was observed in the relapsing-remitting mouse model of EAE.Staining of OPN in bone with the same antibody, MPIIIB10₁, served as apositive control (FIG. 2H). These results show the role of OPN in acute,as well as in relapsing forms of EAE, and indicate that the degree ofexpression of OPN in lesions correlated with the severity of disease.

The role of OPN in demyelinating disease was next tested using OPNdeficient mice (FIGS. 3A-3C) (30). EAE was induced using myelinoligodendrocyte glycoprotein peptide 35-55 (MOG 35-55) in CFA in OPN−/−mice and OPN+/+ controls (31). EAE was observed in 100% of both OPN+/+and OPN−/− mice with MOG 35-55. Despite this, severity of disease wassignificantly reduced in all animals in the OPN−/− group (FIG. 3A), andthese mice were totally protected from EAE-related death (FIG. 3B).Thus, OPN significantly influenced the course of progressive EAE inducedby MOG 35-55.

The rate of relapses and remissions was next tested. During the first 26days, OPN−/− mice displayed a distinct evolution of EAE, with a muchhigher percentage of mice having remissions compared with the controls(FIG. 3C). OPN+/+ and OPN−/− mice were killed on days 28, 48, and 72after immunization for histopathology. Although the clinical courses inthe two groups were quite different, there were similar numbers andappearances of inflammatory foci within the CNS.

To examine whether different immune responses were involved in OPN−/−and OPN+/+ animals, we tested the profile of cytokine expression inthese mice. Because EAE is a T cell-mediated disease, we first analyzedthe T cell proliferative response to the auto-antigen MOG 35-55 in theOPN−/− mice. T cells in OPN−/− mice showed a reduced proliferativeresponse to MOG 35-55, compared with OPN+/+ T cells (FIG. 4A). Inaddition, IL-10 production was increased in T cells reactive to MOG35-55 in OPN−/− mice that had developed EAE, compared with T cells inOPN+/+ mice (FIG. 4B). At the same time, IFN-γ and IL-12 production wasdiminished in the cultures of spleen cells stimulated with MOG (FIGS.4C-4D).

Because IFN-γ and IL-12 are important pro-inflammatory cytokines in MS(1, 33), the finding that in OPN−/− mice there is reduced production ofthese cytokines, is consistent with the notion that OPN plays a criticalrole in the modulation of Th1 immune responses in MS and EAE. Further,IL-10 has been associated with remission from EAE (34). In this context,the enhancement of myelin-specific IL-10 production in OPN−/− mice, mayaccount for the tendency of these mice to go into remission. Sustainedexpression of IL-10 may thus be an important factor in the reversal ofrelapsing MS, and its absence may allow the development of secondaryprogressive MS.

In conclusion our data show that OPN may has pleiotropic functions inthe pathogenesis of demyelinating disease. OPN production by glial cellsmay lead to the attraction of Th1 cells, and its presence in glial andependymal cells may allow inflammatory T cells to penetrate the brain.Finally, our data suggest that neurons may also secrete thisproinflammatory molecule and participate in the autoimmune process.Potentially, neuronal OPN secretion could modulate inflammation anddemyelination, and could influence the clinical severity of the disease.Consistent with this idea, a role for neurons in the pathophysiology ofMS and EAE has recently been described (21,22), and neurons are known tobe capable of cytokine production (35, 36). OPN inhibits cell lysis (6),and thus neuronal OPN might even protect the axon from degenerationduring autoimmune demyelination.

CD44 is a known ligand of OPN, mediating a decrease of IL-10 production(10). As shown here, OPN−/− mice produced elevated IL-10 during thecourse of EAE. We recently demonstrated that anti-CD44 antibodiesprevented EAE (37), suggesting that the proinflammatory effect of OPN inMS and EAE might be mediated by CD44. The binding of OPN to its integrinfibronectin receptor a_(v) b₃ through the arginine-glycine-aspartatetripeptide motif may also perpetuate Th1 inflammation (10). In active MSlesions, the a_(v) subunit of this receptor is overexpressed inmacrophages and endothelial cells, and the b₃ subunit is expressed onendothelial cell luminal surfaces (23). By means of itstripeptide-binding motif, OPN inhibits inducible nitric oxide synthetase(iNOs) (38), which is known to participate in autoimmune demyelination(1). Thus in conclusion, OPN is situated at a number of checkpoints thatwould allow diverse activities in the course of autoimmune-mediateddemyelination.

Methods

Custom Microarrays

Custom microarrays were designed that allow large scale profiling ofmRNA for 517 components of the inflammatory response, includingcytokines, chemokines, various adhesion molecules, and matrixmetalloproteases. Profiles of mRNA transcripts from the spinal cord ofsix Lewis rats with EAE were analyzed. Rats were immunized with 400 mgof guinea pig spinal cord homogenate [GPSCH] and monitored for EAE aspreviously described (12). mRNA was isolated from the brain and spinalcord of three rats with hind limb paralysis (mean EAE score 2.7,indicating severe paraplegia), 15 days after immunization with GPSCH,and from three rats treated with a metalloprotease inhibitor after theinitiation of EAE. It is established that matrix metalloproteaseinhibitors can reverse EAE (13), and rats treated with themetalloprotease inhibitor [RS110379] displayed no clinical disease (meanEAE score 0.2). Spinal cord from two other normal rats served ascontrols.

OPN transcripts were increased 3.4 fold in the spinal cord of rats withEAE and paralysis, compared to controls without EAE (average differencechange for intensity of OPN transcripts was 16609 fluorescent units inuntreated rats with EAE versus an average difference change forintensity in OPN transcripts of 4846 in rats without EAE). Aftertreatment with RS110379, levels of OPN mRNA were no different thancontrol rats without EAE. Thus, there was a 1.1 fold change between theintensity of OPN transcripts in EAE rats treated with MMP inhibitorversus rats without EAE (the average difference of the change inintensity of OPN transcripts on the custom microarrays was 5176 units inrats treated with RS110379 versus an average difference intensity of4846 units in rats without EAE).

Non-Normalized cDNA Brain Libraries

In contrast to normalized libraries in which high frequency transcriptsare preferentially eliminated by nuclease treatment of DNA/RNA hybridsto facilitate detection of rare RNA species, we produced non-normalizedlibraries, where manipulation of clones is avoided. White matter braintissue from the plaques of 3 MS patients was collected and frozen withintwo hours after death. Patient history on the specimen used for thefirst library (herein MS1) included clinically definite MS, and thepresence of active inflammatory lesions. Material for the second MSlibrary (herein MS2) came from a pool of tissues from two patients, onewith acute, active lesions and widespread inflammatory involvement inthe white matter, and the other with chronic, “silent” lesions, withgliosis, but without evidence of a lymphocytic infiltrate. The controllibrary (CTRL) was constructed using pooled mRNA isolated from midbrainwhite matter, inferior temporal cortex, medulla, and posterior parietalcortex tissue removed from a 35-year-old Caucasian male who died fromcardiac failure and who had no neuropathological changes. The librarieswere made in collaboration with Incyte Genomics. Libraries wereconstructed using 1.5 mg of polyA RNA from each sample. cDNA synthesiswas initiated using a NotI-anchored oligo(dT) primer. Double-strandedcDNA was blunted, ligated to EcoRI adaptors, digested with NotI,size-selected, and cloned into the NotI and EcoRI sites of the pTNCYvector (Incyte, Palo Alto, Calif.). Approximately 4,000 clones from eachlibrary were sequenced in ABI automatic DNA Sequencer (AppliedBiosciences, Foster City, Calif.). Annotated data was extracted from theIncyte database LifeSeq Gold® and incorporated into MS Access 2000® andMS Excel 2000® for further analysis. All queries were designed andperformed in MS Access, while charts and tables were generated with MSExcel. Cellular roles were assigned after consulting the Expressed GeneAnatomy Database (EGAD, The Institute for Genomic Research,www.tigr.org/egad). Genomic location was included according to NCBI'sMapView and Genemap'98 (National Institute for BiotechnologyInformation, www.ncbi.nlm.nih.gov). Comparisons of gene frequenciesbetween each MS library and the CTRL were performed and the average foldchange calculated. Differences in gene expression were subjected toFisher's exact test and a P-value of 0.05 or lower, was selected ascriteria for inclusion in each comparison. Results of these experimentsare shown in FIGS. 1A-1C.

We sequenced 3678, 4174, and 3740 clones from MS1, MS2, and controllibraries, respectively. Each of the libraries had a substantial numberof clones with no match to the Genbank database, and were thusconsidered novel. Clones in library MS1 could be assigned to 2387different cDNA species from which 331 corresponded to novel genes. MS2and CTRL yielded 2727 (546 novel) and 2352 (511 novel) speciesrespectively. Analysis of frequency distribution revealed a similarpattern for all three libraries, with the most abundant transcriptsbeing represented by few species including two myelin genes, myelinbasic protein, [MBP], and proteolipid protein, [PLP], and theastrocyte-specific transcript, glial fibrillary acidic protein, [GFAP].Similarly, there was an exponentially decreasing frequency observed forless frequent cDNA species in all three libraries. Taken together thedata reveal that the composition and complexity of the three librarieswere similar, and that there were no obvious biases, therefore enablingcomparative analysis.

Immunostaining

Mice were killed during relapse and remission and perfused with 60 ml of10% formalin. Brain and spinal cord were removed and fixed in the samesolution. Paraffin sections (6-10 μm) were prepared. OPN was detectedwith the monoclonal anti-OPN antibody MPIIIB10₁ (Developmental StudiesHybridoma Bank, Iowa City, Iowa), at 1/50 dilution, using the vectorMouse On Mouse (M.O.M.) immunodetection kit (Vector Laboratories,catalogue no. PK 2200), the Vectastain® Elite ABC kit (VectorLaboratories, catalogue no. PK 6100) according to the manufacturer'sinstructions, and the substrate 3,3′-diaminobenzidine (0.5 mg/ml for 4minutes). The intensity of the cellular staining was evaluated by anobserver blinded to the experimental design according to asemiquantitative scale (three grades). MPIIIB10₁ stains OPN inimmuniohistochemical sections from mice, though it does not recognizeOPN on Western blots (26). The successful use of MPIIIB10₁ in mousesections has been reported (27, 28).

Induction of EAE

We induced EAE with MOG 35-55 in CFA in 129/C57B1/6 OPN−/− mice, and129/C57B1/6 OPN+/+ controls. Here we slightly modified the protocol: weinjected 100 mg of MOG 35-55 emulsion subcutaneously in the flanks ofeach female at day 0, and 400 ng of Pertussis Toxin at day 0 and day 2.Seven OPN+/+ and 6 OPN−/− mice were examined on days 28, 48 and 72 postimmunization for histopathology. Data are unpublished showing similarnumbers and appearances of inflammatory foci within the central nervoussystem in the two groups.

Transcriptional Profiling with Custom Microarrays

Spinal cord was homogenized in TRIzol reagent (Gibco BRL) using aPolytron (Kinematica AG, Switzerland) and total RNA prepared accordingto the recommended protocol. mRNA was purified by two rounds ofselection using oligo(dT) resin (Oligotex, Qiagen). 2 mg of mRNA wasused to prepare double stranded cDNA (Superscript, Gibco BRL). Theprimer for cDNA synthesis contained a T7 RNA polymerase promoter site. 1mg of cDNA was used for an in-vitro transcription reaction (Ambion T7Megascript) with biotinylated CTP and UTP (Enzo Diagnostics, Inc.). Thelabeling procedure amplifies the mRNA population ˜60-fold. Microarraychips (GeneChip™ System, Affymetrix) were hybridized for 16 hours in a45° C. incubator with constant rotation at 60 rpm. Chips were washed andstained on a fluidics station, and scanned using a laser confocalmicroscope. Affymetrix provided the procedures for sample preparation,fluidics station, scanner, and computer workstation. Chips were analysedwith GeneChip v3.1 software, and scaled to a value of 150. The softwaredetermines whether a particular RNA transcript is present or absent,based on the intensity of the signal. Fold change was calculated bydivided the intensity of the average difference change in theexperimental sample by the intensity of the average difference change inthe control.

Example 2

A Method to Treat Multiple Sclerosis and Other Autoimmune Diseases withDNA Encoding Osteonontin

Treatment of mice with DNA encoding murine (i.e., “self protein”)osteopontin induces an anti-osteopontin immunoglobulin response in thehost that inhibits the detrimental impact of osteopontin in perpetuatingthe disease.

DNA encoding murine osteopontin was generated by cloning DNA encodingosteopontin into the pCDNA3 mammalian expression vector. pCDNA3 containsthe CMV promoter and SV-40 large T antigen poly adenylation signal. Thisosteopontin-encoding vector was produced in E. coli and endotoxin-freeDNA was purified using the Qiagen Endo-free Mega-prep kits (Qiagen,Valencia, Calif.).

Mice are injected in the quadricep with 0.1 ml of 0.25% bupivicaine-HCL(Sigma, St. Louis, Mo.) in PBS (0.05 ml per quadricep). Two daysfollowing, mice are injected with 0.05 ml of each “self-plasmid” DNA at1.0 mg/ml in phosphate buffered saline with 0.9 mM calcium in eachquadricep. The plasmid DNA is injected two more times at 2 to 4 weekintervals. The efficacy of osteopontin-encoding self-vector induction ofanti-osteopontin antibodies can be enhanced by co-delivery of CpGimmunostimmulatory oligonucleotides and/or treatment with DNA encodingosteopontin fused to one or more C3d components. Enzyme-linkedimmunosorbent assays were used to monitor levels of anti-osteopontinantibodies, with induction of anti-osteopontin antibodies representingefficacy of the therapy. Mice were subsequently challenged to developEAE with a myelin peptide (typically PLPp139-151) in complete Freund'sadjuvant, and mice pre-treated with self-vector encoding osteoponin havea reduced incidence and severity of EAE as demonstrated in FIG. 5.

Alternatively, strains of mice susceptible to chronic relapsing EAE (forexample, SJL mice) can be induced to develop EAE (for example, withPLPp139-151 in complete Freund's adjuvant) and osteopontin-self-vectortherapy initiated at bi-weekly intervals in mice with established EAE toinduce antibodies against osteopontin to treat the disease. Efficacy ismeasured based on a reduction in the overall disease severity and numberof new episodes of clinical paralysis using standard scoring systems.

In humans with multiple sclerosis, osteopontin-self-vector therapy isinitiated following diagnosis. Efficacy is monitored based on inductionof anti-osteopontin antibodies in the patient with multiple sclerosis,as measured by ELISA analysis. Efficacy is further demonstrated based onthe reduction in the number and size of brain lesions (as measured byMRI scanning), the reduction of the number of disease relapses (episodesof clinical paralysis), and the slowing of progression to disability.

Example 3

Mice were injected with 10 micromolar of cardiotoxin (Sigma) in thetibialis anterior muscle. Five days later mice were given 100 microgramsof plasmid with full length OPN in phosphate buffered saline with 0.9 mMcalcium in the tibialis anterior muscle. The plasmid with OPN wasinjected three more times in intervals of 6-7 days. EAE was induced 7days after the last injection, with myelin oligodendroglial glycoprotein35-55. Controls were performed using no treatment, treatment with PBS ortreatment with the plasmid without the insert. Treatment was assessed ona standard clinical scale relating to disability of the mice. Antibodytiter was also measured in the mice as follows. ELISA plates were coatedwith 50 ng/well of mouse recombinant OPN (R&D Systems Catalog Number:441-OP) at 4 degrees overnight. Plates were washed the second day withPBS and 0.1% Tween (ie PBST Buffer) 3-4 times and then blocked with PBSand 0.25% gelatin for 3 h at 37 degrees. Plates were then washed 5 timeswith PBST and then sera were added in serial dilutions for over nightincubation at 4 degrees (the serum are diluted with PBST 0.25% gelatin).The next day plates were washed with PBST 6 times and goat anti-mouseosteopontin antibody was added (R&D Systems Catalog Number: AF808) atconcentration of 1 ug/ml diluted in PBST 0.25% gelatin and incubate 1 hat 37 degrees Celsius. The plates were then incubated with anti goat Abconjugated to alkaline phosphatase at a dilution of 1:30000 for 1 h. Theplates were wash 6 times and the substrate p-NPP p-nitrophenylphosphatein PBS was added. After development of color, the plates were read at405 nm wavelength.

FIG. 6 shows the results that treatment with the plasmid encoding OPNdiminished clinical symptoms of EAE from day 24 to day 48, p<0.03,during the chronic phase of EAE. FIG. 7 shows that immunization with DNAencoding osteopontin generated an antibody titer that plateaued afterabout 20 days.

REFERENCES

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All publications and patent filings cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

1. A method for treating multiple sclerosis in a patient, the methodcomprising administering to the patient intramuscularly an effectiveamount of a nucleic acid encoding osteopontin, whereby the nucleic acidis expressed in the patient to produce osteopontin, and the osteopontininduces an immune response wherein the immune response comprisesformation of antibodies to osteopontin that reduces the level ofosteopontin in the patient, thereby treating multiple sclerosis.
 2. Themethod of claim 1, wherein the nucleic acid is DNA and further comprisesa promoter and optionally an enhancer in operable linkage to the segmentencoding the osteopontin.
 3. The method of claim 2, wherein the promoteris constitutive.
 4. The method of claim 2, wherein the promoter iscell-type specific.
 5. The method of claim 1, wherein the nucleic acidis DNA.
 6. The method of claim 1, wherein the nucleic acid is RNA. 7.The method of claim 1, wherein the subject is a human.
 8. The method ofclaim 1, further comprising monitoring a decrease in the level ofosteopontin responsive to the administering step.
 9. The method of claim1, wherein the level of osteopontin is monitored in a cell of thepatient selected from the group consisting of a neuron, a macrophage, avascular endothelial cell, an astrocyte and a microglial cell.
 10. Themethod of claim 1, wherein the disorder and the method further comprisesmonitoring a decrease in the symptoms of the patient responsive to theadministering.